Abstract-Na v 1.5, the cardiac isoform of the voltage-gated Na ϩ channel, is critical to heart excitability and conduction. However, the mechanisms regulating its expression at the cell membrane are poorly understood. The Na v 1.5 C-terminus contains a PY-motif (xPPxY) that is known to act as binding site for Nedd4/Nedd4-like ubiquitin-protein ligases. Because Nedd4-2 is well expressed in the heart, we investigated its role in the ubiquitination and regulation of Na v 1.5. Yeast two-hybrid and GST-pulldown experiments revealed an interaction between Na v 1.5 C-terminus and Nedd4-2, which was abrogated by mutating the essential tyrosine of the PY-motif. Ubiquitination of Na v 1.5 was detected in both transfected HEK cells and heart extracts. Furthermore, Nedd4-2-dependent ubiquitination of Na v 1.5 was observed. To test for a functional role of Nedd4-2, patch-clamp experiments were performed on HEK cells expressing wild-type and mutant forms of both Na v 1.5 and Nedd4-2. Na v 1.5 current density was decreased by 65% upon Nedd4-2 cotransfection, whereas the PY-motif mutant channels were not affected. In contrast, a catalytically inactive Nedd4-2 had no effect, indicating that ubiquitination mediates this downregulation. However, Nedd4-2 did not alter the whole-cell or the single channel biophysical properties of Na v 1.5. Consistent with the functional findings, localization at the cell periphery of Na v 1.5-YFP fusion proteins was reduced upon Nedd4-2 coexpression. The Nedd4-1 isoform did not regulate Na v 1.5, suggesting that Nedd4-2 is a specific regulator of Na v 1.5. These results demonstrate that Na v 1.5 can be ubiquitinated in heart tissues and that the ubiquitin-protein ligase Nedd4-2 acts on Na v 1.5 by decreasing the channel density at the cell surface.
Serum- and glucocorticoid-regulated kinase 1 (SGK1) is an aldosterone-regulated early response gene product that regulates the activity of several ion transport proteins, most notably that of the epithelial sodium channel (ENaC). Recent evidence has established that SGK1 phosphorylates and inhibits Nedd4-2 (neural precursor cell-expressed, developmentally down-regulated protein 4-2), a ubiquitin ligase that decreases cell surface expression of the channel and possibly stimulates its degradation. The mechanistic basis for this SGK1-induced Nedd4-2 inhibition is currently unknown. In this study we show that SGK1-mediated phosphorylation of Nedd4-2 induces its interaction with members of the 14-3-3 family of regulatory proteins. Through functional characterization of Nedd4-2-mutant proteins, we demonstrate that this interaction is required for SGK1-mediated inhibition of Nedd4-2. The concerted action of SGK1 and 14-3-3 appears to disrupt Nedd4-2-mediated ubiquitination of ENaC, thus providing a mechanism by which SGK1 modulates the ENaC-mediated Na(+) current. Finally, the expression pattern of 14-3-3 is also consistent with a functional role in distal nephron Na(+) transport. These results demonstrate a novel, physiologically significant role for 14-3-3 proteins in modulating ubiquitin ligase-dependent pathways in the control of epithelial ion transport.
The mineralocorticoid hormone aldosterone controls sodium reabsorption and BP largely by regulating the cell-surface expression and function of the epithelial sodium channel (ENaC) in target kidney tubules. Part of the stimulatory effect of aldosterone on ENaC is mediated by the induction of serum-and glucocorticoid-regulated kinase 1 (Sgk1), a kinase that interferes with the ubiquitylation of ENaC by ubiquitin-protein ligase Nedd4-2. In vivo early aldosterone-regulated mRNA now has been identified in microselected mouse distal nephron by microarray. From 22 mRNA that displayed a two-fold or more change, 13 were downregulated and nine were upregulated. Besides Sgk1, the induced mRNA include Grem2 (protein related to DAN and cerebrus [PRDC]), activating transcription factor 3, cAMP responsive element modulator, and the ubiquitin-specific protease Usp2-45. The induction of this last enzyme isoform was verified in mouse distal nephron tubule at the protein level. With the use of Hek293 cells, Xenopus oocytes, and mpkCCD c14 cells as expression systems, it was shown that Usp2-45 deubiquitylates ENaC and stimulates ENaC-mediated sodium transport, an effect that is not additive to that of Sgk1. A deubiquitylating enzyme that targets ENaC in vitro and thus may play a role in sodium transport regulation was identified within a series of new in vivo early aldosterone-regulated gene products.
Aldosterone plays a central role in Na؉ homeostasis by controlling Na ؉ reabsorption in the aldosterone-sensitive distal nephron involving the epithelial Na ؉ channel (ENaC). Part of the effects of aldosterone is mediated by serum and glucocorticoid-induced kinase 1 (Sgk1), a Ser/Thr kinase whose expression is rapidly induced by aldosterone and that increases in heterologous expression systems ENaC cell surface abundance and activity. Previous work in Xenopus laevis oocytes suggested that Sgk1 phosphorylates specific residues (Ser212 and Ser328) on the ubiquitin-protein ligase Nedd4-2, an enzyme that directly interacts with ENaC and negatively controls channel density at the plasma membrane. It further indicated that phosphorylation of Nedd4-2 led to impairment of ENaC/Nedd4-2 interaction and consequently to more channels at the cell surface. These data suggested a novel mode of aldosterone-dependent action, yet this was not demonstrated formally in epithelial cells that physiologically express ENaC. Here it is shown, with the use of an anti-phospho-Ser328-mNedd4-2 antibody, that 2 to 6 h of aldosterone treatment induces an increase in Nedd4-2 phosphorylation, both in a mouse cortical collecting duct cell line (mpkCCD cl4 ) and in kidneys of adrenalectomized rats. This augmentation, which is accompanied by a raise in Sgk1 expression and transepithelial Na ؉ transport, is sensitive to phosphatidylinositol-3 kinase inhibition, as is Sgk1 phosphorylation and Na ؉ transport. Hence, these data provide evidence in cortical collecting duct cells in vitro and in vivo that Sgk1-dependent phosphorylation of Nedd4-2 is part of the aldosterone response.
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